Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of the interleukin 10-encoding (IL-10) mRNA by ovine (ov-) cells, in response to mitogenic stimulation, was assessed by Northern blot and polymerase chain reaction (PCR) analyses using a human (hu) IL-10 cDNA probe and oligodeoxyribonucleotide primers based on homologous regions of the human and murine IL-10 cDNA sequences. A 315-bp cDNA generated by the PCR analysis was cloned and used to screen a lipopolysaccharide-stimulated alveolar ov-macrophage cDNA library. The full-length ov-cDNA sequence isolated translates to a protein of 177 amino acids (aa) with a predicted 18-aa leader sequence and molecular mass of 20,165 Da. Expression in a mammalian system demonstrated that the ov-cDNA encoded a protein with the expected IL-10 biological activity. Both recombinant huIL-10 and supernatants from COS cells transfected with an expression vector containing the ovIL-10 cDNA inhibited production of IL-1 and tumour necrosis factor-alpha by ov-alveolar macrophages. Genomic DNA analysis indicated ovIL-10 exists as a single gene within the ov-genome.
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PMID:Cloning and characterisation of an ovine interleukin-10-encoding cDNA. 762 47

Calmodulin-dependent nitric-oxide synthase, with an apparent molecular mass of 125 kDa, was induced in the liver of rats treated with Propionibacterium acnes and Escherichia coli lipopolysaccharide. Clones were isolated from a cDNA library obtained from induced rat liver using oligonucleotide probes which were synthesized based on the amino acid sequences of peptides of the purified enzyme. Four overlapping cDNA clones for a 3.8-kbp region were isolated and the nucleotide sequences were determined. These clones encompassed an open-reading frame of 3441 bases encoding 1147 amino acids. The deduced amino acid sequence of the cDNA suggested that the protein contains binding sites for NADPH, FAD and FMN. The structure of the possible calmodulin-binding site, consisting of a strongly hydrophobic region surrounded by basic amino acids, is present. The full-length cDNA was expressed in COS 1 cells under the control of a cytomegalovirus promoter and the expressed enzyme was found to be a calmodulin-dependent nitric-oxide synthase. A structural comparison suggested that the liver nitric-oxide synthase is the same as the macrophage enzyme. Northern-blot analysis showed that the mRNA in the liver is approximately 4.2 kb long and is induced transcriptionally by treatment with P. acnes and lipopolysaccharide.
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PMID:Molecular cloning of a cDNA encoding an inducible calmodulin-dependent nitric-oxide synthase from rat liver and its expression in COS 1 cells. 769 62

Rat prostaglandin endoperoxidase synthase-2 (PGHS-2) cDNA was cloned from rat calvarial osteoblasts total RNA by RT-PCR. The primary sequence of rat PGHS-2 had 98% and 92% identity to the mouse and human enzymes, respectively. Transfection of the rat PGHS-2 cDNA into COS 7 cells, followed by the addition of 20 microM arachidonic acid, resulted in a dramatic increase in PGE2 released from these cells. The amount of PGE2 produced was comparable to that obtained from cells similarly transfected with human PGHS-1 cDNA. In the rat paw carrageenin-oedema inflammatory model, the injected paw had elevated levels of PGHS-2 mRNA compared to the control paw. In a rat pyrexia model, injection of the pyrogen lipopolysaccharide, resulted in elevated levels of PGHS-2 mRNA in the brain. These results suggest that PGHS-2 expression plays a role both in inflammation and fever.
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PMID:Cloning and expression of rat prostaglandin endoperoxide synthase (cyclooxygenase)-2 cDNA. 791 14

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.
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PMID:Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells. 864 64

Interferons (IFN) and lipopolysaccharide (LPS) cause multiple changes in isoprenoid-modified proteins in murine macrophages, the most dramatic being the expression of a prenyl protein of 65 kDa. The guanylate binding proteins (GBPs) are IFN-inducible GTP-binding proteins of approximately 65 kDa that possess a CaaX motif at their C-terminus, indicating that they might be substrates for prenyltransferases. The human GBP1 protein, when expressed in transfected COS-1 cells, incorporates radioactivity from the isoprenoid precursor [3H]mevalonate. In addition, huGBPs expressed from the endogenous genes in IFN-gamma-treated human fibroblasts or monocytic cells were also found to be isoprenoid modified. IFN-gamma-induced huGBPs in HL-60 cells were not labeled by the specific C20 isoprenoid, [3H]geranylgeraniol, but did show decreased isoprenoid incorporation in cells treated with the farnesyl transferase inhibitor BZA-5B, indicating that huGBPs in HL-60 cells are probably modified by a C15 farnesyl rather than the more common C20 lipid. Differentiated HL-60 cells treated with IFN-gamma/LPS showed no change in the profile of constitutive isoprenylated proteins and the IFN-gamma/LPS-induced huGBPs remained prenylated. Despite being prenylated, huGBP1 in COS cells and endogenous huGBPs in HL-60 cells were primarily (approximately 85%) cytosolic. Human GBPs are thus among the select group of prenyl proteins whose synthesis is tightly regulated by a cytokine. HuGBP1 is an abundant protein whose prenylation may be vulnerable to farnesyl transferase inhibitors that are designed to prevent farnesylation of Ras proteins.
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PMID:Prenylation of an interferon-gamma-induced GTP-binding protein: the human guanylate binding protein, huGBP1. 883 Aug

The induction of immunoglobulin heavy chain (IgH) 3' enhancer activity has been coupled to ligand/receptor-dependent activation of resting B cells. To search for transcriptional target sites that account for this induction, extracts from lipopolysaccharide (LPS)-stimulated B cells and cell lines were used. Here we describe, by gel-retardation analysis, the identification of an NF-kappaB site and an adjacent nuclear factor ets-like (NFE) site in the 3' enhancer. The NFE motif binds four protein complexes in resting B cell extracts, of which two are down-regulated upon LPS stimulation. Gel shift-shift experiments of the NF-kappaB complexes with specific antibodies identified p50 and c-Rel proteins to be the predominant factors in primary LPS-stimulated cell extracts. Site-directed mutagenesis of these motifs demonstrates that they contribute to part of the enhancer activity in plasma cells. One copy of the NFkappaB/NFE motifs, linked to a heterologous reporter construct, displays lymphoid-restricted reporter gene activity in transient transfection assays. Mutation of either site abrogates all promoter activity. Complementation experiments demonstrate that although p50 and c-Rel expression vectors reconstitute transcription of an intact NF-kappaB/NFE reporter construct in a dose-dependent manner, mutation of the NFE site or the NF-kappaB site abrogates essentially all transcriptional activity in both plasma cells and in COS cells. Taken together, we provide evidence for the existence of an activator, NFE, which in combination with the p50 and c-Rel proteins, are part of the transcription factor machinery that regulates 3' enhancer activity, and thus the control of the IgH locus in late B lymphocyte development.
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PMID:NFE, a new transcriptional activator that facilitates p50 and c-Rel-dependent IgH 3' enhancer activity. 904 19

Platelet-activating factor (PAF) is a potent phospholipid with diverse physiological and pathological actions, and it is inactivated by PAF acetylhydrolase. In this study, we analyzed the tissue distribution of the plasma PAF acetylhydrolase mRNA in humans. We isolated a 3.5-kilobase fragment containing the 5' genomic sequence of the plasma PAF acetylhydrolase gene and further characterized the promoter activity. We determined the transcriptional initiation site by primer extension. We then prepared constructs containing various lengths of 5' genomic fragments fused to a luciferase reporter gene and transfected these constructs into COS-7 cells. We found that there is more than one region in the 1.3-kilobase 5' genomic sequence conferring promoter activity and that a very short 5'-flanking region (72 base pairs) is sufficient for more than 65% of the basal activity. In parallel, we examined the regulation of expression of the PAF acetylhydrolase gene. We found that interferon-gamma (IFNgamma) and lipopolysaccharide (LPS) significantly inhibited synthesis of PAF acetylhydrolase, whereas other cytokines, including IFNalpha, interleukin (IL) 1alpha, IL4, IL6, tumor necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor, had a smaller or no effect in human monocyte-derived macrophages. Furthermore, transfection of the promoter/reporter construct into macrophage RAW264.7 cells revealed that IFNgamma and LPS decreased the promoter activity by 35% and 50%, respectively, whereas PAF stimulated it by 52% via its receptor. The promoter activity was much lower in monocytic U937 cells compared with the basal level in COS-7 cells, while the activities in P388D1 and RAW264.7 macrophagic cells were considerably higher than the basal level in COS-7 cells. There are multiple regions in the PAF acetylhydrolase promoter that contain responsive elements for signal transducer and activators of transcription-related proteins, and also for myeloid-specific transcription factors. Our data indicate that the opposite of mRNA expression in monocytes versus macrophages is due to inhibition of the promoter activity in the former and activation in the latter cells.
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PMID:Expression of plasma platelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of inflammation. 946 91

Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to those of snake venom metalloproteinases and disintegrins. The ADAMTS-1 gene encodes a new type of ADAM protein with respect to possessing the thrombospondin (TSP) type I motifs. Expression of the gene is induced in kidney and heart by in vivo administration of lipopolysaccharide, suggesting a possible role in the inflammatory reaction. In this study, we characterized the ADAMTS-1 gene product by using a transient expression system in COS-7 cells. We found that the precursor and processed forms of ADAMTS-1 were secreted from cells. Under normal growth conditions, little or none of both forms was detected in the cell culture medium, and instead the majority was found associated with the extracellular matrix (ECM). In addition, when cells were cultured in the presence of heparin, the mature form of ADAMTS-1 protein was detected in the cell culture medium, suggesting that binding of ADAMTS-1 to the ECM is mediated through sulfated glycosaminoglycans such as heparan sulfate. Analyses of deletion mutants of the ADAMTS-1 protein revealed that the spacer region as well as three TSP type I motifs in the carboxyl-terminal region of the ADAMTS-1 protein are important for a tight interaction with the ECM. These results suggest that the ADAMTS-1 is a unique ADAM family protein that anchors at the ECM.
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PMID:ADAMTS-1 protein anchors at the extracellular matrix through the thrombospondin type I motifs and its spacing region. 959 39

We have isolated a full-length cDNA for an inducible nitric oxide synthase (iNOS) from guinea-pig lung. The cDNA has a 3447 bp open reading frame encoding 1149 amino acid residues. The deduced amino acid sequence is approx. 80% identical with iNOS of human epithelial cells and murine macrophages. Consensus recognition sites for cofactors are highly conserved. COS cell lysate transfected with the guinea-pig iNOS shows significant levels of nitric oxide synthase (NOS) activity, and this is inhibited by 79% by chelation of Ca2+ ions. The NOS activity is restored in a concentration-dependent manner by increasing the free Ca2+ level. The NOS activity is also inhibited by trifluoperazine, a calmodulin antagonist, which suggests that the Ca2+ dependence is due to Ca2+-dependent calmodulin binding to the enzyme. Northern blot analysis reveals that the cloned iNOS mRNA is expressed in the lung and the colon in normal guinea pigs. Stimulation in vivo by lipopolysaccharide induces the expression of iNOS in the kidney, the spleen and the colon, but in the lung the same stimulation decreases its expression. These results suggest that the cloned guinea-pig iNOS is distinct in characteristics and expression from previously described iNOS forms.
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PMID:Molecular cloning and characterization of Ca2+-dependent inducible nitric oxide synthase from guinea-pig lung. 967 42

Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta). Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography. Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.
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PMID:A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein. 999 Mar 17


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